Lipid rafts, microdomain heterogeneity and inter-organelle contacts: impacts on membrane preparation for proteomic studies.
In plasma membrane proteome research, contamination of the isolated plasma membrane fraction with proteins from other organelles is still a problem. Even if highly specific isolation methods are used, such as density gradient centrifugation combined with selective extraction, contaminating proteins cannot be completely removed. To solve this problem, a protocol for the isolation of highly pure plasma membrane fractions from rat liver and two different hepatocellular carcinoma cell lines was developed. Magnetic beads with immobilized mAb's against highly expressed membrane proteins were used for specific binding of membrane vesicles and their separation from other organelles. Isolated plasma membranes were further selectively solubilized with different reagents and analyzed by use of different methods, such as Western blotting, 1- and 2-DE, and MS. Purification and further selective solubilization was validated by use of mAb's against the marker integral plasma membrane protein carcinoembryonic antigen cell adhesion molecule 1, and identification of isolated proteins by MS. The method presented here minimizes contamination with other organelles and enables further identification of membrane proteins.